Actinomycetes

Dr.K.Sivakumar
CentreofAdvancedStudyinMarineBiology
AnnamalaiUniversity


ctinomycetesarebestknownfortheirabilitytoproduceantibiotics
and are gram positive bacteria which comprise a group of
branching unicellular microorganisms. They produce branching
mycelium which may be of two kinds viz., substrate mycelium and
aerial mycelium. Among actinomycetes, the streptomycetes are the
dominant. The nonstreptomycetes are called rare actinomycetes,
comprisingapproximately100genera.
Members of the actinomycetes, which live in marine
environment, are poorly understood and only few reports are available
pertaining to actinomycetes from mangroves (Siva Kumar, 2001;
Vikineswari et al. 1997; Rathana Kala & Chandrika, 1993;
Lakshmanaperumalsamy,1978).
IsolationofActinomycetesfromSediments
Foractinomycetes,thesamplescanbecollectedbyinsertingapolyvinyl
corer(10cmdia.)(previouslysterilizedwithalcohol)intothesediments.
The corer is sterilized with alcohol before sampling at each station. The
central portion of the top 2 cm sediment sample can be taken out with
the help of a sterile spatula. This sample can be transferred to a sterile
polythene bag and transported immediately to the laboratory. The
sediment samples thus collected are airdried aseptically. After a week,
the sediment samples are to be incubated at 55
0
C for 5 min
(Balagurunathan, 1992). Then, 10fold serial dilutions of the sediment
samplesshouldbeprepared,usingfilteredandsterilized50%seawater.
OnemloftheseriallydilutedsamplesshouldbeplatedintheKusters
Agar (Siva Kumar, 2001) in triplicate petriplates. To minimize fungal
contamination,allagarplatesshouldbesupplementedwith50ug/mlof
nystatin.Theactinomycetecoloniesthatappearonthepetriplatescanbe
counted from 5
th
day onwards, upto 28
th
day. All the colonies that are
growingonthepetriplatescanbeseparatelystreakedinpetriplates,sub
cultured,ensuredfortheiraxenicityandmaintainedinslants.
A


198 Actinomycetes

Identification
Various approaches for the identification of actinomycets are given
brieflybelow:
a)MolecularApproach
The most powerful approaches to taxonomy are through the study of
nucleicacids.Becausetheseareeitherdirectgeneproductsorthegenes
themselves and comparisons of nucleic aids yield considerable
informationabouttruerelatedness.
Molecular systematics, which includes both classification and
identification, has its origin in the early nucleic acid hybridization
studies, but has achieved a new status following the introduction of
nucleic acid sequencing techniques (ODonnell et al., 1993). Significance
of phylogenetic studies based on 16S rDNA sequences is increasing in
thesystematicsofbacteria andactinomycetes(Yokota,1997).Sequences
of 16S ribosomal DNA have provided actinomycetologists with a
phylogenetic tree that allows the investigation of evolution of
actinomycetesandalsoprovidesthebasisforidentification.
Analysis of the 16S rDNA begins by isolating DNA (Hapwood,
1985) and amplifying the gene coding for 16S rRNA using the
polymerase chain reaction (e.g. Siva Kumar, 2001). The purified DNA
fragmentsaredirectlysequenced.Thesequencingreactionsareperformed
using DNA sequencer in order to determine the order in which the bases
are arranged within the length of sample (Xu LiHua, et al., 1999) and a
computeristhenusedforstudyingthesequenceforidentificationusing
phylogenetic analysis procedures. However, analysis of 16S rDNA
generallyallowsustoidentifytheorganismsuptothegenuslevelonly.
b)ChemotaxonomicalApproach
Chemotaxonomyisthestudyofchemicalvariationinorganismsandthe
use of chemical characters in the classification and identification. It is
oneofthevaluablemethodstoidentifythegeneraofactinomycetes.
Studies of Cummins and Harris (1956) established that
actinomyceteshaveacellwallcompositionakintothatofgrampositive
bacteria, and also indicated that the chemical composition of the cell
wall might furnish practical methods of differentiating various types of
actinomycetes. This is because of the fact that chemical components of
the organisms that satisfy the following conditions, have significant
meaninginsystematics.



K. Sivakumar 199
i. They should be distributed universally among the
microorganismsstudied;and,
ii. The components should be homologous among the strains
withinataxon,whilesignificantdifferencesexistbetweenthe
taxatobedifferentiated.
Presence of Diaminopimelic Acid (DAP) isomers is one of the
most important cellwall properties of grampositive bacteria and
actinomycetes. Most bacteria have a characteristic wall envelope,
composed of peptidoglycan. The 2, 6 Diaminopimelic Acid (DAP) is
widely distributed as a key aminoacid and it has optical isomers. The
systematicsignificanceliesmostlyinthekeyaminoacidwithtwoamino
bases, and determination of the key aminoacid is usually sufficient for
characterisation.IfDAPispresent,bacteriagenerallycontainoneofthe
isomers, the LLform or the mesoform, mostly located in the
peptidoglycan.
Majorconstituentsofcellwallofactinomycetes(Lechevalierand
Lechevalier,1970)areasfollows:

Cellwall glycine mesoDAP LLDAP

I + +
II + +**
III +
**hydroxyDAP(mayalsobepresent)
The sugar composition often provides valuable information on
theclassificationandidentificationofactinomycetes.Actinomycetecells
contain some kinds of sugars, in addition to the glucosamine and
muramicacidofpeptidoglycan.Thesugarpatternplaysakeyroleinthe
identification of sporulating actinomycetes which have mesoDAP in
their cell walls. However, the actinomycetes which have LLDAP along
withglycine(wallchemotypeI)havenocharacteristicpatternofsugars
(Lechevalier and Lechevalier, 1970) and hence the whole cell sugar test
hasnotreceivedmuchattentionhere.

c)ClassicalApproach
Classical approaches for classification make use of morphological,
physiological, and biochemical characters. The classical method


200 Actinomycetes

described in the identification key by Nonomura (1974) and Bergeys
Manual of Determinative Bacteriology (Buchanan & Gibbons, 1974) is
very much useful in the identification of streptomycetes. These
characteristics have been commonly employed in taxonomy of
streptomycetes for many years. They are quite useful in routine
identification.Theyareasfollows.

1.AerialMassColour
The colour of the mature sporulating aerial mycelium is recorded in a
simple way (White, grey, red, green, blue and violet). When the aerial
mass colour falls between two colour series, both the colours are
recorded. If the aerial mass colour of a strain to be studied shows
intermediatetints,thenalso,boththecolourseriesarenoted.

2.MelanoidPigments
The grouping is made on the production of melanoid pigments (i.e.
greenishbrown,brownishblackordistinctbrown,pigmentmodifiedby
other colours) on the medium. The strains are grouped as melanoid
pigmentproduced(+)andnotproduced().

3.ReverseSidePigments
The strains were divided into two groups, according to their ability to
produce characteristic pigments on the reverse side of the colony,
namely, distinctive (+) and not distinctive or none (). In case, a colour
withlowchromasuchaspaleyellow,oliveoryellowishbrownoccurs,it
isincludedinthelattergroup().

4.SolublePigments
The strains are divided into two groups by their ability to produce
soluble pigments other than melanin: namely, produced (+) and not
produced (). The colour is recorded (red, orange, green, yellow, blue
andviolet).

5.SporeChainMorphology
With regard to spore chains, the strains can be grouped into sections.
The species belonging to the genus Streptomyces are divided into three
sections (Shirling & Gottlieb, 1966), namely rectiflexibiles (RF),
retinaculiaperti(RA)andSpirales(S).Whenastrainformstwotypesof
sporechains,botharenoted(e.g.SRA).



K. Sivakumar 201

Characteristics of the sporebearing hyphae and spore chains

should be determined by using direct microscopic examination of the
culture surface. Adequate magnification (400x) could be used to
establish the presence or absence of spore chains and to observe the
natureofsporophores.
Spore morphological characters of the strains can be studied by
inoculating a loopful of one week old cultures into 1.5% agar medium
contained in test tubes at 37
0
C. The actinomycete should be suspended
andthoroughlymixedinthesemisolidagarmediumand1or2dropsof
the medium could be aseptically pipetted on to a sterile glass slide. A
drop of agarshould be spread well onthe slide and allowed to solidify
into a thin film so as to facilitate direct observation under microscope.
Theculturesshouldbeincubatedat28+2
0
Candexaminedperiodically
for the formation of aerial mycelium, sporophore structure and spore
morphology.

6.SporeSurface
Sporemorphologyanditssurfacefeaturesshouldbeobservedunderthe
scanning electron microscope. The cross hatched cultures prepared for
observation under the light microscope can be used for this purpose.
RFSporechains(400X)
RA Spore chains (400X)
SpiralSporechains(400X)


202 Actinomycetes

Theelectrongridshouldbecleanedandadhesivetapeshouldbeplaced
on the surface of the grid. The mature spores of the strain should be
carefully placed on the surface of the adhesive tape and gold coating
should be applied for half an hour and the specimen can be examined
under the electron microscope at different magnifications. The spore
silhouettescanbecharacterizedassmooth,spiny,hairyandwarty.

7.AssimilationofCarbonSource
The ability of different actinomycete strains in utilizing various carbon
compoundsassourceofenergyshouldbestudiedfollowingthemethod
recommended by International Streptomyces Project (Shirling and
Gottlieb, 1966). Chemically, pure carbon sources, certified to be free of
admixturewithothercarbohydratesorcontaminatingmaterials,should
beusedforthispurpose.Carbonsourcesforthistestcouldbearabinose,
xylose, inositol, mannitol, fructose, rhamnose, sucrose and raffinose.
These carbon sources should be sterilized by ether sterilization without
heating.
Comparing the properties of the isolated strain with the
representative species found in the key of Nonomura (1974) and
BergeysManualofDeterminativeBacteriology(BuchananandGibbons,
1974) can help in the species level identification. If the isolated strain
couldnotbeassignedtoanyofthevalidrepresentativeslistedinthekey
ofNonomura(1974)andBergeysManualofDeterminativeBacteriology
(Buchanan & Gibbons, 1974), then it can be identified based on the
numericaltaxonomicstudies.

d)NumericalTaxonomicApproach
Numerical taxonomy involves examining many strains for a large
number of characters prior to assigning the test organism to a cluster
based on shared features. The numerically defined taxa are polythetic;
so, no single property is either indispensable or sufficient to entitle an
organism for membership of a group. Once classification has been
achieved, clusterspecific or predictive characters can be selected for
identification(Williamsetal,1983).
Numerical taxonomy was first applied to Streptomyces by
Silvestri et al. (1962). The numerical taxonomic study of the genus
StreptomycesbyWilliamsetal.(1983)involvesdeterminationof139unit
charactersfor394typeculturesofStreptomyces;clustersweredefinedat
77.5%or81%Ssmand63%Sjsimilaritylevels,andtheformercoeffieient



K. Sivakumar 203
is being used to define the clusters. His study includes 23 major, 20
minorand25singlememberclusters.
The numerical classification of the genus Streptomyces by
Kampfer et al. (1991) involves determination of 329 physiological tests.
His study includes 15 major clusters, 34 minor clusters and 40 single
member clusters which are defined at 81.5% similarity level Ssm using
the simple matching coefficient (Sokal and Michener, 1958) and 59.6 to
64.6% similarity level Sj using Jaccard coefficient (Sneath, 1957). Thus,
numerical taxonomy provides us with an invaluable framework for
Streptomycestaxonomy,includingidentificationofspecies.
Preservation
The preservation methods are similar to that of bacteria such as sub
culturing, freezing especially in liquid nitrogen, freezedrying and
maintenanceofstrainsinmineraloil.

Conclusion
Studies on actinomycetes are very limited and the actinomycetes have
been mentioned incidentally, on the microbial community of marine
habitats. Further, only little information is available on the
actinomycetes of the mangrove environment (which is one among the
mostproductivecoastalecosystems)withregardtotheiroccurrenceand
distribution (Vikineswari et al., 1997; Rathna Kala and Chandrika, 1993;
Lakshmanaperumalsamy,1978).

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